Expression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori.

Objective(s) : Helicobacter pylori, a human specific gastric pathogen is a causative agent of chronic active gastritis. The vacuolating cytotoxin (VacA) is an effective virulence factor involved in gastric injury. The aim of this study was to construct a recombinant protein containing antigenic region of VacA gene and determine its antigenicity. Materials and Methods: The antigenic region of VacA gene was detected by bioinformatics methods. The polymerase chain reaction method was used to amplify a highly antigenic region of VacA gene from chromosomal DNA of H. pylori. The eluted product was cloned into the prokaryotic expression vector pET32a. The target protein was expressed in the Escherichia coli BL21 (DE3) pLysS. The bacteria including pET32a-VacA plasmids were induced by IPTG. The antigenicity was finally studied by western blotting using sera of 15 H. pylori infected patients after purification. Results: Enzyme digestion analysis, PCR and DNA sequencing results showed that the target gene was inserted correctly into the recombinant vector. The expressed protein was purified successfully via affinity chromatography. Data indicated that antigenic region of VacA protein from Helicobacter pylori was recognized by all 15 patient’s sera. Conclusion : Our data showed that antigenic region of VacA protein can be expressed by in E. co.li. This protein was recognized by sera patients suffering from H. pylori infection. the recombinant protein has similar epitopes and close antigenic properties to the natural form of this antigen. Recombinant antigenic region of VacA protein also seems to be a promising antigen for protective and serologic diagnosis .


Introduction
Helicobacter pylori is a Gram negative curved microaerophilic bacilli colonized in the human gastric mucosa and establishes a chronic infection that is tightly associated with atrophic gastritis, peptic and duodenal ulcers, gastric adenocarcinoma and gastric MALT (Mucosa Associated Lymphoid Tissue) lymphoma (1,2). It was recently defined as a group 1 carcinogen (3). Moreover, It is estimated that 60% of the world's population are infected with H. pylori, ranging from 20-30% in developed countries up to 70-80% in developing countries (4,5). An important virulence factor of H. pylori is a secreted toxin known as vacuolating cytotoxin (VacA) that induces the formation of intracellular vacuoles in epithelial cells. This protein causes multiple alterations in human cells including cell vacuolization, depolarization of membrane potential, membrane channel formation, alteration of mitochondrial membrane permeability, apoptosis, disruption of endosomal/lysosomal function, activation of mitogen-activated protein kinases, inhibition of antigen presentation, and inhibition of T-cell activation and proliferation (6)(7)(8)(9). Recent molecular and cellular studies of VacA action have shown that it is a major virulence factor that is involved in the pathogenesis of inflammation in H. pylori-induced gastritis and ulceration (10,11). This protein is a proper candidate for vaccines. In order to stimulate more specific immune response * PCR amplification was performed in a 50 μl total volume containing 3 μl of template DNA, 2 μl of each primers (10 picomole), 8 μl MgCl₂ (25 mM), 1.5 μl dNTP mixture, 5 μl PCR buffer (10X) and 1 μl of expand DNA polymerase (Roche, Germany). The following conditions were used for amplification: initial denaturation at 94°C for 5 min followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 53°C for 1 min and extension at 72°C for 1 min and further extension for 5 min at 72°C (15). The PCR product was analyzed by horizontal agarose gel electrophoresis in 1X TBE buffer and visualized by ethidium bromide staining on UV transilluminator.

Cloning of VacA gene in bacterial expression vector and transformation
The PCR product was purified from the agarose gel by high pure PCR product purification kit (Roche, Germany) according to the manufacturer guideline. It was digested with EcoRI and XhoI, and inserted into the expression vector pET32a (+) (Novagene, USA) containing N-terminal histidine tag (6His.tag) which was digested by corresponding restriction endonuclease enzymes using T4 DNA ligase enzyme (Cinagen, Iran) at 22°C during 1 hr incubation. It was afterwards identified by restriction enzyme digestion and PCR. E. coli DH5α and E. coli BL21 (DE3) pLysS competent cells were prepared by calcium chloride (CaCl₂) method (15) and were used for transformation of plasmid. The plasmid pET32a-VacA was transformed into competent E. coli DH5α (Stratagene, USA) as the primary host in order for amplification of recombinant plasmid and subsequently into E. coli BL21 (DE3) pLysS (Stratagene, USA) cells as the expression host for recombinant protein production using ampicillin and chloramphenicol resistance for selection (15). The sequence of inserted fragment was analyzed by dideoxy chain termination procedure as described by Sanger et al (16).

Gene expression, purification
The recombinant E. coli BL21 (DE3) pLysS was grown in 2 ml nutrient broth medium being supplemented with ampicillin (100 mg/ml) and chloramphenicol (35 mg/ml) on shaking incubator for overnight at 37°C. On the next day, 500 μl of culture was inoculated in 50 ml of Nutrient Broth medium (0.5 g yeast extract, 1 g Bacto pepton, 0.

Western Blot analysis
The integrity of the product was confirmed by western blot analysis. Western blotting was performed according to the standard protocol (15) using sera from a 15 H. pylori sero-positive patients

Statistical analysis
The statistical analysis of data was performed using SPSS software (version 18: SPSS Inc., Chicago). Also used of chi-square test.

Detection of antigenic region
The result obtained from the three abovementioned servers was in accordance; therefore, nucleotide sequence of 1224 till 2457 was selected as region with high antigenic properties ( Figure 1).  Expression and purification of recombinant protein pET32a-VacA was introduced into E. coli BL21 (DE3) pLysS and the expression of recombinant protein was induced by IPTG. A band in the range of molecular weight ~65 KDa was found in SDS-PAGE analysis that was similar to what had been predicted ( Figure 3). Due to the presence of 6His.tag at the Nterminal region of rVacA, purified recombinant protein was obtained using Ni-NTA resin in an affinity chromatography procedure (Figure 3). The concentration of highly expressed recombinant protein was calculated and 2.1 mg/ml was obtained by further purification and dialysis.

Immunoblotting analysis
In order for determining the antigenicity of recombinant antigenic fragment of VacA protein in H. pylori positive sera of patients, identity confirmation of rVacA was applied via western blotting and the desired band was observed with an estimated molecular weight of ~65 KDa. All 15 H. pylori infected sera were interacted with recombinant VacA protein, whereas all 15 H. pylori negative human sera failed to act similarly. The specific interaction between patient's sera and purified rVacA protein is shown in Figure 4. The H. pylori negative sera of patients were used as negative control.

Discussion
In the present study, we have shown that recombinant antigenic region of VacA protein can be produced by pET32a (+) in E. coli. Our data indicate that antigenic protein of VacA of H. pylori could be detected by all 15 patient's sera; therefore, this recombinant VacA can be used for detecting anti-VacA antibody.
Currently, the main treatment to eliminate H. pylori is combined antibiotics therapy. However, due to incomplete eradication of H. pylori through antibiotic treatment, the cost of combination therapy, re-infection, patients' complaints and emerges of antibiotic resistance strains, vaccination could be suggested as a preventive approach (17).
The VacA has been approved as an effective virulence factor involved in the pathogenesis of H. pylori infection (12,18,19). Thus, this protein can be considered as a vaccine candidate.
VacA is initially encodes as a 140-KDa precursor protein that subsequently undergoes both Nterminal and C-terminal processing to yield an 87-95-KDa mature secreted toxin (20, 21). Mature secreted VacA toxin yields two fragments that are 33 and 55 KDa (p33 and p55) (22). The N-terminal p33 domain contains a hydrophobic sequence involved in pore formation, and the p55 domain contains cellbinding domains (23).
Mature secreted VacA assembles into a large flowerlike oligomer in the extracellular space. The oligomer is disassembled by exposure to acid or alkaline conditions. The VacA monomer associates with forming anion-selective membrane channels (11).
The intact protein contains full-length DNA sequence and unneeded epitopes and due to the large size of gene fragment, can result in a higher molecular weight, which makes it difficult to clone, express and purify; hence, limits this protein to be used in whole sequence as a vaccine candidate. In this study, in order to increase the immunogenicity and decrease the ineffective immune response, part of VacA gene with suitable antigenic properties, which had been determined with bioinformatics methods, was used for detecting the antibody. Therefore, the protein obtained in our study is smaller than normal VacA protein with the same antigenic properties.  (25).
In order for giving a high-level expression of recombinant protein, we used E. coli BL21 (DE3) pLysS due to cytoplasmic protease deficiency in this strain (26).
Expression of protein in pET system added several amino acids such as thioredoxin protein (109 amino acids) (Trx.tag) and 6His.tag and S.Tag sequences to recombinant peptide that is beneficial for detection and purification of target protein through affinity chromatography procedure. These additional amino acids increase the molecular weight from 7900 to 28000 of synthesized peptide (27)